Method for inhibiting tumor invasion or spreading in a subject

ABSTRACT

The present invention provides for a method for inhibiting tumor invasion or metastasis in a subject which comprises administering to the subject a therapeutically effective amount of a form of soluble Receptor for Advanced Glycation Endproducts (RAGE). The present invention also provides a method for evaluating the ability of an agent to inhibit tumor invasion in a local cellular environment which comprises: (a) admixing with cell culture media an effective amount of the agent; (b) contacting a tumor cell in cell culture with the media from step (a); (c) determining the amount of spreading of the tumor cell culture, and (d) comparing the amount of spreading of the tumor cell culture determined in step (c) with the amount determined in the absence of the agent, thus evaluating the ability of the agent to inhibit tumor invasion in the local cellular environment. The present invention also provides a pharmaceutical composition which comprises a therapeutically effective amount of the agent evaluated in the aforementioned method and a pharmaceutically acceptable carrier.

The invention disclosed herein was made with Government support underNational Institutes of Health Grant No. AG00602 from the Department ofHealth and Human Services. Accordingly, the U.S. Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byauthor and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding Sequence Listing and the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

An important means by which tumors grow and invade surrounding normaltissue is by a complex series of cell—cell and cell matrix interactions.We have focused on the interaction of tumor cells with matrix-associatedcomponents. The Receptor to AGE (RAGE) interacts with a range ofphysiologically and pathophysiologically-relevant ligands (1-5). Innormal developing neurons of the central nervous system, the expressionof RAGE is markedly enhanced and co-localizes with that of its ligand,amphoterin. Amphoterin, a matrix-associated polypeptide, is expressed indeveloping neurons and certain tumor cells, such as rat C6 glioma cells(6-12).

SUMMARY OF THE INVENTION

The present invention provides for a method for inhibiting tumorinvasion or metastasis in a subject which comprises administering to thesubject a therapeutically effective amount of a form of soluble Receptorfor Advanced Glycation Endproducts (RAGE). The present invention alsoprovides a method for evaluating the ability of an agent to inhibittumor invasion in a local cellular environment which comprises: (a)admixing with cell culture media an effective amount of the agent; (b)contacting a tumor cell in cell culture with the media from step (a);(c) determining the amount of spreading of the tumor cell culture, and(d) comparing the amount of spreading of the tumor cell culturedetermined in step (c) with the amount determined in the absence of theagent, thus evaluating the ability of the agent to inhibit tumorinvasion in the local cellular environment. The present invention alsoprovides a pharmaceutical composition which comprises a therapeuticallyeffective amount of the agent evaluated in the aforementioned method anda pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B. Transfection of C6 glioma cells with constructs expressingsRAGE results in diminished tumor volume compared with mock-transfectedtumor cells. 1×10⁵ tumor cells/ml (0.1 ml) were injected into the backsof nude mice. Cells were previously transfected either with constructsoverexpressing sRAGE or mock-transfection (vector alone) as describedabove. At the indicated days after tumor injection, tumor volume wasmeasured. The mean±standard error of the mean is reported. Twenty micewere studied per experimental group. Statistical considerations areindicated. This experiment was repeated three times with analogousresults.

FIG. 2A-2B. The growth rate of sRAGE-transfected tumor cells is notdifferent than that of mock-transfected tumor cells, in vivo. The rateof tumor cell growth was calculated and the slope of the line determined(±standard error) using Microsoft Excel. The y axis represents thenatural logarithm of the tumor volume and the x axis represents time(days). These studies indicated that the slope of the growth curve formock-transfected tumor cells was 0.247±0.025. For sRAGE-transfectedtumor cells, the slope of the growth curve was 0.241±0.029.

FIG. 3A-3B. The rate of growth of sRAGE-transfected tumor cells issimilar to that of mock-transfected tumor cells in in vitro studies. C6glioma cells transfected with either sRAGE or mock were placed into 96well tissue culture wells. On day three after onset of incubation, cellswere fixed and stained and relative cell number assessed using theCYQUANT® assay from AMERSHAM®. These studies indicated that the relativecell number of sRAGE-transfected C6 cells on day three was 0.90±0.03 and1.0±0.04 in mock-transfected C6 tumor cells (p=0.11).

FIG. 4A-4B. Systemic administration of sRAGE results in diminished tumorvolume in C6 glioma cells injected into nude mice. C6 glioma cells(1×10⁵/ml; 0.1 ml) were injected into the backs of nude mice.Immediately after injection, once daily injections of either mouse sRAGE(20 or 2 μg/day) or mouse serum albumin (MSA; 40 μg/day) were begunintraperitoneally. Tumor volume was measured on days 14 and 17 afterinjection. For each experimental group, n-10. Statistical considerationsare indicated.

FIG. 5A-5B. The rate of growth of C6 glioma cells is similar in sRAGEand MSA-treated mice in vivo. The rate of tumor cell growth wascalculated and the slope of the line determined (±standard error) usingMicrosoft Excel. The y axis represents the natural logarithm of thetumor volume and the X axis represents time (days). The slope of thegrowth curve for C6 glioma tumor cells grown in the presence of mouseserum albumin was 0.264±0.03. The slopes of the growth curve for C6glioma cells grown in the presence of sRAGE (20 vs. 2 μ/ml) were notdifferent (0.311±0.04 and 0.313±0.04, respectively).

FIG. 6A-6B. The rate of growth of tumor cells is similar in the presenceof either sRAGE or mouse serum albumin in in vitro studies. C6 gliomacells were placed into 96 well tissue culture wells and grown in thepresence of either sRAGE (20 or 2 μg/ml) or mouse serum albumin (40μg/ml). On day three after onset of incubation, cells were fixed andstained and relative cell number assessed using the CYQUANT® assay fromAMERSHAM®. Relative cell number (day three) of cells treated with MSAwas 1.0±0.045. Similarly, relative cell number in the presence of sRAGE(20 or 2 μg/ml) was 1.03±0.06 and 1.02±0.06, respectively. Statisticalconsiderations are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a method for inhibiting tumorinvasion or metastasis in a subject which comprises administering to thesubject a therapeutically effective amount of a form of soluble Receptorfor Advanced Glycation Endproducts (RAGE).

As used herein, a “form of soluble RAGE” encompasses peptides which arederived from naturally occurring RAGE protein. The following areexamples of forms of soluble RAGE: mature human soluble RAGE, maturebovine soluble RAGE, mature murine soluble RAGE, fragments or portionsof a soluble RAGE. Representative peptides of the present inventioninclude but are not limited to peptides having an amino acid sequencewhich corresponds to amino acid numbers (2-30), (5-35), (10-40),(15-45), (20-50), (25-55), (30-60), (30-65), (10-60), (8-100), 14-75),(24-80), (33-75), (45-110) of human sRAGE protein. In one embodiment theform of soluble RAGE may consist essentially of any such portion of thehuman RAGE peptide. In another embodiment, the form of soluble RAGE mayconsist essentially of a peptide which comprises any such portion of thehuman RAGE amino acid having the sequence from amino acid at position 1(alanine) to amino acid number 332 (alanine) (without considering the 22amino acid leader sequence).

The abbreviations used herein for amino acids are those abbreviationswhich are conventionally used: A=Ala=Alanine; R=Arg=Arginine;N=Asn=Asparagine; D=Asp=Aspartic acid; C=Cys=Cysteine; Q=Gln=Glutamine;E=Glu=Gutamic acid; G=Gly=Glycine; H=His=Histidine; I=Ile=Isoleucine;L=Leu=Leucine; K=Lys =Lysine; M=Met=Methionine; F=Phe=Phenyalanine;P=Pro=Proline; S=Ser=Serine; T=Thr=Threonine; W=Trp=Tryptophan;Y=Tyr=Tyrosine; V=,Val=Valine. The amino acids may be L- or D- aminoacids. An amino acid may be replaced by a synthetic amino acid which isaltered so as to increase the half-life of the peptide or to increasethe potency of the peptide, or to increase the bioavailability of thepeptide.

In one embodiment the form of soluble RAGE comprises a peptide havingthe sequence from alanine at position 1 to alanine at position 332 ofhuman RAGE.

In another embodiment the form of soluble RAGE comprises a peptide of10-40 amino acids having a sequence taken from within the sequence fromalanine at position 1 to alanine at position 332 of human RAGE.

In another embodiment, the form of soluble RAGE comprises a peptidehaving the following sequence of mature human RAGE. The following is theamino acid sequence of mature human RAGE (which does not include a 22amino acid leader sequence):

(Seq I.D. No.1). Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu Val LeuLys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp Lys Leu AsnThr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser Pro Gln Gly Gly Gly Pro TrpAsp Ser Val Ala Arg Val Leu Pro Asn Gly Ser Leu Phe Leu Pro Ala Val GlyIle Gln Asp Glu Gly Ile Phe Arg Gys Gln Ala Met Asn Arg Asn Gly Lys GluThr Lys Ser Asn Tyr Arg Val Arg Val Tyr Gln Ile Pro Gly Lys Pro Glu IleVal Asp Ser Ala Ser Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr CysVal Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp His Leu Asp Gly LysPro Leu Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu Gln Thr Arg Arg HisPro Glu Thr Gly Leu Phe Thr Leu Gln Ser Glu Leu Met Val Thr Pro Ala ArgGly Gly Asp Pro Arg Pro Thr Phe Ser Cys Ser Phe Ser Pro Gly Leu Pro ArgHis Arg Ala Leu Arg Thr Ala Pro Ile Gln Pro Arg Val Trp Glu Pro Val ProLeu Glu Glu Val Gln Leu Val Val Glu Pro Glu Gly Gly Ala Val Ala Pro GlyGly Thr Val Thr Leu Thr Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile HisTrp Met Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro Val Leu Ile LeuPro Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr His SerSer His Gly Pro Gln Glu Ser Arg Ala Val Ser Ile Ser Ile Ile Glu Pro GlyGlu Glu Gly Pro Thr Ala Gly Ser Val Gly Gly Ser Gly Leu Gly Thr Leu AlaLeu Ala Leu Gly Ile Leu Gly Gly Leu Gly Thr Ala

In one embodiment the form of soluble RAGE comprises a V domain ofnaturally occuring soluble RAGE. In another embodiment the form ofsoluble RAGE comprises a C domain of naturally occurring soluble RAGE.The 22 amino acid leader sequence of immature human RAGE is Met Ala AlaGly Thr Ala Val Gly Ala Trp Val Leu Val Leu Ser Leu Trp Gly Ala Val ValGly (SEQ ID NO:2).

In one embodiment the form of soluble RAGE is a peptide expressed by areplicable vector containing nucleic acid encoding the form of solubleRAGE. In one embodiment the form of soluble RAGE is a peptide whichcorresponds to all or part of soluble RAGE expressed by a replicablevector containing nucleic acid encoding the form of soluble RAGE. Inanother embodiment the replicable vector is capable of expressing thepeptide within a tumor cell in a subject.

In another embodiment the tumor cell is a eukaryotic cell.

In another embodiment the replicable vector is a plasmid, an attenuatedvirus, a phage, a phagemid or a linear nucleic acid.

In another embodiment, the method further comprises administering apharmaceutically acceptable carrier to the subject during theadministration of the form of soluble RAGE.

In one embodiment the administration is via intralesional,intraperitoneal, intramuscular or intravenous injection; infusion;intrathecal administration; subcutaneous administration;liposome-mediated delivery; or topical, nasal, oral, ocular or oticdelivery. As used herein, intrathecal administration includesadministration in the cerebrospinal fluid by lumbar puncture.

In one embodiment of the present invention, the subject is a mammal. Inanother embodiment, the mammal is a human.

In one embodiment, the form of soluble RAGE is administered daily,weekly or monthly. The form of soluble RAGE may be delivered hourly,daily, weekly, monthly, yearly (e.g. in a time release form) or as a onetime delivery. The delivery may be continuous delivery for a period oftime, e.g. intravenous delivery. The agent or pharmaceutical compositionof the present invention may be delivered intercranially or into thespinal fluid. In another embodiment, the therapeutically effectiveamount comprises a dose from about 0.000001 mg/kg body weight to about100 mg/kg body weight. In a preferred embodiment, the therapeuticallyeffective amount comprises a dose of from about 100 ng/day/kg bodyweight to about 200 mg/day/kg body weight.

The present invention also provides a method for evaluating the abilityof an agent to inhibit tumor invasion in a local cellular environmentwhich comprises: (a) admixing with cell culture media an effectiveamount of the agent; (b) contacting a tumor cell in cell culture withthe media from step (a); (c) determining the amount of spreading of thetumor cell culture, and (d) comparing the amount of spreading of thetumor cell culture determined in step (c) with the amount determined inthe absence of the agent, thus evaluating the ability of the agent toinhibit tumor invasion in the local cellular environment.

In one embodiment the tumor cell is a eukaryotic cell. In anotherembodiment the tumor cell is a cell of a subject.

In a further embodiment, the subject is a human, a mouse, a rat, a dogor a non-human primate.

In a further embodiment, the agent comprises a peptide, apeptidomimetic, a nucleic acid, a synthetic organic molecule, aninorganic molecule, a carbohydrate, a lipid, an antibody or fragmentthereof, or a small molecule. The antibody may be a monoclonal antibody.The antibody may be a polyclonal antibody. In one embodiment, thefragment of the antibody comprises a Fab fragment. In another embodimentthe fragment of the antibody comprises a complementarity determiningregion or a variable region. The agent may be conjugated to a carrier.The peptide or agent may be linked to an antibody, such as a Fab or a Fcfragment for specifically targeted delivery.

In one embodiment the peptide is a synthetic peptide or a peptideanalog. The peptide may be a non-natural peptide which has chirality notfound in nature, i.e. D- amino acids or L-amino acids. In anotherembodiment the peptide comprises at least a portion of thesequence-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-(Seq. I.D. No. 3).

In a further embodiment the peptide comprises at least a portion of thesequence -Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met- (Seq. I.D. No.4). In another embodiment the peptide has the amino acid sequenceA-Q-N-I-T-A-R-I-G-E-P-L-V-L-K-C-K-G-A-P-K-K-P-P-Q-R-L-E-W-K (Seq. I.D.No. 5).

In another embodiment the peptide has the amino acid sequenceA-Q-N-I-T-A-R-I-G-E (Seq. I.D. No. 6).

In addition to naturally-occurring forms of soluble RAGE, the presentinvention also embraces other peptides such as peptide analogs of sRAGE.Such analogs include fragments of sRAGE. Following the procedures of thepublished application by Alton et al. (WO 83/04053), one can readilydesign and manufacture genes coding for microbial expression of peptideshaving primary conformations which differ from that herein specified forin terms of the identity or location of one or more residues (e.g.,substitutions, terminal and intermediate additions and deletions).Alternately, modifications of cDNA and genomic genes can be readilyaccomplished by well-known site-directed mutagenesis techniques andemployed to generate analogs and derivatives of SRAGE polypeptide. Suchproducts share at least one of the biological properties of sRAGE butmay differ in others. As examples, products of the invention includethose which are foreshortened by e.g., deletions; or those which aremore stable to hydrolysis (and, therefore, may have more pronounced orlongerlasting effects than naturally-occurring); or which have beenaltered to delete or to add one or more potential sites forO-glycosylation and/or N-glycosylation or which have one or morecysteine residues deleted or replaced by e.g., alanine or serineresidues and are potentially more easily isolated in active form frommicrobial systems; or which have one or more tyrosine residues replacedby phenylalanine and bind more or less readily to target proteins or toreceptors on target cells. Also comprehended are polypeptide fragmentsduplicating only a part of the continuous amino acid sequence orsecondary conformations within sRAGE, which fragments may possess oneproperty of sRAGE and not others. It is noteworthy that activity is notnecessary for any one or more of the polypeptides of the invention tohave therapeutic utility or utility in other contexts, such as in assaysof sRAGE antagonism. Competitive antagonists may be quite useful in, forexample, cases of overproduction of sRAGE.

The agent of the present invention may be a peptidomimetic which may beat least partially unnatural. The agent may be a small molecule mimic ofa portion of the amino acid sequence of sRAGE. The agent may haveincreased stability, efficacy, potency and bioavailability by virtue ofthe mimic. Further, the agent may have decreased toxicity. Thepeptidomimetic agent may have enhanced mucosal intestinal permeability.The agent may be synthetically prepared. The agent of the presentinvention may include L-,D- , DL- or unnatural amino acids, alpha,alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid (anisoelectronic analog of alanine). The peptide backbone of the agent mayhave at least one bond replaced with PSI-[CH═CH] (Kempf et al. 1991).The agent may further include trifluorotyrosine, p-Cl-phenylalanine,p-Br-phenylalanine, poly-L-propargylglycine, poly-D,L-allyl glycine, orpoly-L-allyl glycine. Examples of unnatural amino acids which may besuitable amino acid mimics include β-alanine, L-α-amino butyric acid,L-Υ-amino butyric acid, L-α-amino isobutyric acid, L-∈-amino caproicacid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, cysteine(acetamindomethyl), N-∈-Boc-N-α-CBZ-L-lysine, N-∈-Boc-N-α-Fmoc-L-lysine,L-methionine sulfone, L-norleucine, L-norvaline,N-α-Boc-N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine,Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, Boc-L-thioproline.(Blondelle, et al. 1994; Pinilla, et al. 1995).

In one embodiment of the present invention the agent is a form ofsoluble human RAGE. In another embodiment the agent is an extracellularportion human RAGE. In another embodiment the agent inhibits aninteraction between the tumor cell and an extracellular matrix molecule.In one embodiment the extracellular matrix molecule is a laminin, afibronectin, amphoterin, a cadherin, an integrin or a hyaluronic acid.In another embodiment the integrin is an αVβV integrin, an αVβIIIintegrin, or an αIβII integrin. In another embodiment, the laminin is β1laminin. In another embodiment the agent inhibits binding of RAGE toamphoterin.

The present invention also provides a pharmaceutical composition whichcomprises a therapeutically effective amount of the agent evaluated inthe aforementioned method and a pharmaceutically acceptable carrier. Inone embodiment the carrier is a diluent, an aerosol, a topical carrier,an aqueous solution, a replicable nucleic acid vector, a liposome, amagnetic bead, a nonaqueous solution or a solid carrier.

The actual effective amount will be based upon the size of the subject.The biodegradability of the agent, the bioactivity of the agent and thebioavailability of the agent are factors which will alter the effectiveamount. The agent may be delivered topically in a creme or salvecarrier. It may be reapplied as needed based upon the absorbancy of thecarrier to the skin or mucosa or wound. If the agent does not degradequickly, is bioavailable and highly active, a smaller amount will berequired to be effective. The effective amount will be known to one ofskill in the art; it will also be dependent upon the form of the agent,the size of the agent and the bioactivity of the agent. One of skill inthe art could routinely perform empirical activity tests for a agent todetermine the bioactivity in bioassays and thus determine the effectiveamount.

The present invention also provides a method for inhibiting tumorinvasion or metastasis in a subject which comprises administering to thesubject a therapeutically effective amount of the aforementionedpharmaceutical composition.

One embodiment of the present invention is a composition which comprisesa form of soluble RAGE and a pharmaceutically acceptable carrier. Inanother embodiment the invention provides a composition which comprisesthe aforementioned agent and a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, acetate buffered saline solution(a likely vehicle for parenteral administration), water, emulsions suchas an oil/water emulsion or a triglyceride emulsion, various types ofwetting agents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

When administered orally or topically, such agents and pharmaceuticalcompositions would be delivered using different carriers. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid, talc, vegetable fats or oils, gums,glycols, or other known excipients. Such carriers may also includeflavor and color additives or other ingredients. The specific carrierwould need to be selected based upon the desired method of deliver,e.g., PBS could be used for intravenous or systemic delivery andvegetable fats, creams, salves, ointments or gels may be used fortopical delivery.

Such compositions are liquids or lyophilized or otherwise driedformulations and include diluents of various buffer content (e.g.,Tris-HCl., acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents(e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzylalcohol, parabens), bulking substances or tonicity modifiers (e.g.,lactose, mannitol), covalent attachment of polymers such as polyethyleneglycol to the agent, complexation with metal ions, or incorporation ofthe agent into or onto particulate preparations of polymeric agents suchas polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes,micro emulsions, micelles, unilamellar or multi lamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance of the agent or composition. The choice ofcompositions will depend on the physical and chemical properties of theagent.

The agent of the present invention may be delivered locally via acapsule which allows sustained release of the agent or the peptide overa period of time. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines) and the agent coupled toantibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors. Otherembodiments of the compositions of the invention incorporate particulateforms protective coatings, protease inhibitors or permeation enhancersfor various routes of administration, including parenteral, pulmonary,nasal and oral.

When administered, agents (such as a peptide comprising the V-domain ofsRAGE) are often cleared rapidly from the circulation and may thereforeelicit relatively short-lived pharmacological activity. Consequently,frequent injections of relatively large doses of bioactive agents may byrequired to sustain therapeutic efficacy. Agents modified by thecovalent attachment of water-soluble polymers such as polyethyleneglycol, copolymers of polyethylene glycol and polypropylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline are known to exhibit substantiallylonger half-lives in blood following intravenous injection than do thecorresponding unmodified agents (Abuchowski et al., 1981; Newmark etal., 1982; and Katre et al., 1987). Such modifications may also increasethe agent's solubility in aqueous solution, eliminate aggregation,enhance the physical and chemical stability of the agent, and greatlyreduce the immunogenicity and reactivity of the agent. As a result, thedesired in vivo biological activity may be achieved by theadministration of such polymer-agent adducts less frequently or in lowerdoses than with the unmodified agent.

Attachment of polyethylene glycol (PEG) to agents is particularly usefulbecause PEG has very low toxicity in mammals (Carpenter et al., 1971).For example, a PEG adduct of adenosine deaminase was approved in theUnited States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The agent of the present invention may be delivered in amicroencapsulation device so as to reduce or prevent an host immuneresponse against the agent or against cells which may produce thecompound. The agent of the present invention may also be deliveredmicroencapsulated in a membrane, such as a liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS EXAMPLE 1 Inhibition of Tumor Growth and Spread bySoluble Receptor for Age (sRAGE) in C6-glioma Tumors in Nude Mice

An important means by which tumors grow and invade surrounding normaltissue is by a complex series of cell—cell and cell matrix interactions.We have focused on the interaction of tumor cells with matrix-associatedcomponents. The Receptor to AGE (RAGE) interacts with a range ofphysiologically and pathophysiologically-relevant ligands (1-5). Innormal developing neurons of the central nervous system, the expressionof RAGE is markedly enhanced and co-localizes with that of its ligand,amphoterin. Amphoterin, a matrix-associated polypeptide, is expressed indeveloping neurons and certain tumor cells, such as rat C6 glioma cells(6-12).

In in vitro experiments, the interaction of neuronal RAGE withamphoterin mediates neurite outgrowth: on amphoterin-coated matrices,neurite outgrowth is inhibited in the presence of either anti-RAGE(Fab)₂ or soluble RAGE (sRAGE; the extracellular two thirds of RAGE). Incontrast, blocking access to RAGE had no effect on neurite outgrowth onlaminin- or poly-lysine-coated matrices. As in the case of amphoterin,the expression of RAGE decreases after birth in developing rats.Notably, however, the expression of amphoterin is also increased intumor cells. It has been postulated that in that context, amphoterin mayeffect matrix degradation by activation of plasminogen: events criticalin the ability of tumors to locally invade their immediate environment.Indeed, the ability of tumors to modulate local tissue and vasculatureis essential for distal invasion and the development of metastases.

In our studies, we tested the hypothesis that blockade of tumor cellRAGE would interfere with the ability of tumors to compromise theintegrity of their local environment, at least in part by disruption ofthe RAGE (cellular)-amphoterin (matrix) interaction. We demonstrate herethat when C6 (rat) glioma cells were transfected with constructsdesigned to overexpress soluble RAGE (sRAGE) in the local tumorenvironment, tumor growth was significantly diminished in nude mice. Incontrast, mock transfection of C6 glioma cells (vector only) had noeffect on tumor growth compared with untreated cells. Similarly, when C6glioma cells were injected into nude mice, intraperitonealadministration of sRAGE resulted in dose-dependent diminution of tumorsize. In contrast, treatment with mouse serum albumin was withouteffect.

Taken together, our studies indicate that administration of sRAGE may bean important means by which to limit tumor growth in the localenvironment and, likely, the development of distant metastases. Ourstudies indicate that interruption of cell (RAGE)-matrix (amphoterinand/or similar structures) is at least one mechanism by which sRAGElimits tumor growth. Our data strongly suggest, however, that sRAGE doesnot affect the rate of tumor cell growth.

These studies indicate that RAGE may be a novel target for theprevention of local tumor growth and invasion. Soluble RAGE (sRAGE) maythus represent a model structure for the development of agents to limittumor growth and invasion into the local environment, and, potentially,the development of distant metastases.

Materials and Methods

Cell culture. C6 glioma cells (rat) we obtained from the American TypeTissue Corporation (ATCC, Rockville, Md.) and characterized aspreviously described (Singh et al, 1997). Cells were grown asrecommended by the ATCC. Transfection of constructs expressing eitherhuman soluble RAGE (Neeper et al., 1992) were prepared and transfectedinto C6 glioma cells using LipoFECTAMINE® (Life Technologies, GrandIsland, N.Y.). Stable lines were selected using G418 (LifeTechnologies). Constructs containing vector alone (mock) weretransfected into C6 cells as controls.

Animal studies. NCR nude mice were obtained from Taconic Laboratories.At the age of 8-10 weeks, C6 glioma cells (usually about 10⁵ cells/ml)were injected into the lower backs of the mice. Tumor volume wasmeasured sequentially and recorded. At sacrifice, mice were deeplyanesthetized and then tumors removed and weighed.

Preparation of mouse soluble RAGE. Murine soluble RAGE was preparedusing a baculovirus-Sf9 cell expression system. Murine sRAGE waspurified from cellular supernatant using FPLC Mono S®(Amersham-Pharmacia®) and endotoxin removed using De-Toxigel® columns(Pierce, Rockford, Ill.). The final product was pure based on SDS-PAGEelectrophoresis (nonreducing conditions). No detectable endotoxin wasfound in the final samples (limulus amebocyte assay. Sigma Chemical Co.,St. Louis, Mo.). Control mice were treated with mouse serum albumin(MSA: Sigma Chemical Co.)

Injection of sRAGE into mice. Mice were treated immediately uponinjection of the tumor cells into the back of the mouse with theindicated dose of sRAGE or mouse serum albumin (equimolar concentration)through the day of sacrifice. Protein was injected intraperitoneally ina volume of 0.1 ml in phosphate-buffered saline.

Quantitation of cell number. C6 glioma cells were grown onto the wellsof 96-well plates. On day, cells were fixed and relative cell numberdetermined using the CYQUANT® assay system from Amersham.

Results

We first determined by RT-PCR that C6 glioma cells expressed mRNA forRAGE; the presence of RAGE antigen was confirmed byimmunohistochemistry. In order to determine if sRAGE might interrupttumor growth and invasion, we performed two sets of experiments. In thefirst, C6 glioma cells were transfected with constructs to expresseither sRAGE or vector alone. In other experiments, C6 glioma cells wereinjected into nude mice; tumors were generated, and then mice weretreated parenterally with either sRAGE or mouse serum albumin.

sRAGE-transfected C6 Glioma Cells

sRAGE-transfected or mock-transfected C6 glioma cells were injected intonude mice and tumor volume sequentially measured. When tumor volume wasmeasure on days 14, 17 and 21, substantially smaller tumors wereobserved in sRAGE-transfected cells compared with mock-transfected tumorcells (FIG. 1). On day 14, mean tumor volume in nude mice injected withmock-transfected tumors was 315,276.5±3,790.4 μm³ vs 27,875.4±4,199.6μm³ mean tumor volume in sRAGE-transfected tumor (11.3-fold difference;p<0.00005). On day 17, mean tumor volume in nude mice injected withmock-transfected tumor was 823,759±142,841.5 μm³ vs. 50,645±5,109.7 μm³(16.2-fold difference; p<0.00005). On day 21, mean tumor volume in nudemice injected with mock-transfected tumor was 2,200,000±420,235 μm³ vs.152,000±22,487.3 μm³ (14.5-fold difference; p=0.00001). When evaluatedby ELISA, an at least three-fold increase in levels of sRAGE were foundin sRAGE-transfected tumors vs. Those transfected with vector alone(mock). Histologic analysis confirmed markedly smaller tumors, withevidence of necrosis.

The growth rate of the transfected tumor cells (sRAGE and mock) derivedfrom analysis of the above in vivo data revealed that there was nodifference between the rate of growth of sRAGE-transfected CΔ gliomacells compare with those mock-transfected (FIG. 2). When the natural logof tumor volume was plotted vs time, the slope of the growth curve formock-transfected tumor cells the slope of the growth curve was0.241±0.025. These data are consistent with the concept that sRAGE doesnot alter cellular replication. Rather, they suggest that theinteraction of tumor cells with their local environment is affected in amanner inconsistent with their local spread and invasion.

To further confirm that transfection of sRAGE into C6 glioma cells didnot alter the rate of tumor growth, we demonstrated that whensRAGE-transfected or mock transfected C6 glioma cells were grown inculture, no difference in the cell number was observed at three daysincubation (FIG. 3). Using a colormetric assay system from Amersham forquantitation of cell number, on day three, relative cell number ofsRAGE-transfected C6 cells was 0.90±0.03 vs. 1.0 ±0.04 inmock-transfected C6 tumor cells (p=0.11). These data were furthersuggestive of the importance of sRAGE in limiting tumor cell growth andspread into the local tumor environment in vivo and suggest that sRAGEdoes not impact on cellular replication processes.

Systemic Administration of sRAGE

In order to further delineate the role of sRAGE in limiting tumor growthand invasion into the local environment, C6 tumors were established inNCR nude mice by injection of 10⁵ cells/ml (0.1 ml) C6 glioma cells(without transfection). Immediately after injection of tumor cells,intraperitoneal administration of sRAGE was begun (either 20 or 2μg/day) or mouse serum albumin (FIG. 4). On day 14, mean tumor volume inMSA-treated mice was 459,000±87,457.6 μm³. In mice treated with sRAGE,20 μg/day, however, mean tumor volume was decreased 2-fold(225,000±38,054 μm³; p=0.03). In contrast, mice treated with sRAGE, 2μg/day, demonstrated no significant change in tumor volume(305,000±79,998; p=0.21 [when compared with those treated with MSA]}. Onday 17, mean tumor volume in MSA-treated mice was 935,000±117,499 μm³.In contrast, mice treated with sRAGE, 2 μg/day, demonstrated nosignificant change in tumor volume (637,000±127,698 μm³; p=0.22 [whencompared with those treated with MSA]).

When the growth rate of the tumor cells was plotted, no apparentdifferences were noted among tumors in mice treated with sRAGE (bothdoses) or mouse serum albumin (FIG. 5). When the natural log of thetumor volume was plotted vs. time, the slope of the growth curve for C6glioma tumor cells grown in the presence of mouse serum albumin was0.264±0.03. The slopes of the growth curve for C6 glioma cells grown inthe presence of sRAGE (20 vs. 2 μg/ml) were not different (0.311±0.04and 0.313±0.04, respectively) These data further support the hypothesisthat sRAGE does not impact on cellular replication; rather, sRAGEappears to inhibit cellular interaction with matrix components that isnecessary for tumor impingement in the local environment.

Finally, when C6 cells were cultured in the presence of sRAGE (high andlow doses; 20 and 2 μg/ml) or mouse serum albumin, no apparentdifference in relative cell number was observed (FIG. 6). Relative cellnumber (day three) of cells treated with MSA was 1.0±0.045. Similarly,relative cell number in the presence of sRAGE (20 or 2 μg/ml) was1.03±0.06 and 1.02±0.06, respectively. These data further support theconcept that sRAGE does not impact upon cellular proliferation; rather,it more likely affects the ability of tumor cells in vivo to grow andextend into their local environment.

Discussion

A number of factors have been suggested to be important in the abilityof tumors to grow and invade their local environment. Central to theseinvolves processes such as angiogenesis, the ability of the tumors todevelop tumor-specific vasculature; matrix degradation, in which theability of matrix metalloproteinases (MMPs) to degrade the surroundingmatrix is critical for the local spread and invasiveness of tumors;inhibition of the action of tissue inhibitors of metalloproteinases(TIMPs), whose natural function is to inhibit the activity of MMPs; andintegrin-cell/integrin/matrix interactions, reactions in which specificligand-receptor contact initiates signaling cascades that eventuate intumor spread (14-25).

Certainly, a multitude of factors have been postulated to result inlocal tumor growth and invasion. In the present studies, the ability ofsRAGE to result in diminished tumor volume appears to result not from adirect effect of sRAGE on cellular replication processes. Rather, theinteraction of tumor cells with their local environment appearshampered. We postulated that perhaps one means by which this occurs isby inhibition of the tumor RAGE-matrix amphoterin components. Amphoterinand likely related structures have been suggested to activateplasminogen, thereby effecting one means by which, for example, MMPs maybe activated in the tumor environment. It is quite possible, however,that additional, as yet unidentified mechanisms exist by which sRAGEexerts beneficial effects in this model.

A current focus of this work is to study the effects of sRAGE in a modelof tumor metastases and distal invasion. At this time, studies areunderway utilizing Lewis lung carcinoma cells; a model in which distantinvasion of the tumor into the pleura and lung parenchyma ensues afterremoval of the primary lesion (placed onto the lower back of the mice).

In conclusion, while precise mechanisms underlying the beneficialeffects of sRAGE are under study in this model of local tumor growth,our data suggest that sRAGE may represent a novel structure in thedesign of agents to limit tumor spread and invasion. In this context,sRAGE may also exert beneficial effects in the prevention of tumordistal growth and metastases.

REFERENCES

1. Schmidt, A -M., et al. J. Biol. Chem. 276: 14987-14997, 1992.

2. Neeper, M., et al. J. Biol. Chem. 267: 14998-15004, 1992.

3. Hori, O., et al. J. Biol. Chem. 170: 25752-25761, 1995.

4. Yan, S. D., et al. Nature 382: 685-691, 1996.

5. Yan, S. D., et al. Proc. Natl. Acad. Sci. 94: 5296-5301, 1997.

6. Schmidt, A -M., et al. In Hormones and Signalling, Volume I, AcademicPress, p. 41-63, 1997.

7. Rauvala, H. and Pihlaskari, R. J. Biol. Chem. 262: 16625-16635, 1987.

8. Merenmies, J., et al. J. Biol. Chem. 266: 16722-16729, 1991.

9. Salmivirta, M., et al. Exp. Cell Res. 200: 444-451, 1992.

10. Mohan, P S, J., et al. Bioc. And Biophyx. Res. Comm. 182: 689-696,1992.

11. Parkkinen, J., J. Biol. Chem. 268: 19726-19738, 1993.

12. Parkkinen, J. and H. Rauvala. J. Biol. Chem. 266: 16730-16735, 1991.

13. Singh, S. P., et al. Neuroreport 8: 2359-2363, 1997.

14. Price, J. T., et al. Critical Reviews in Biochemistry and MolecularBiology. 32: 175-253, 1997.

15. Timar, J., et al. Anticancer Research 16(6A): 3299-3306, 1997.

16. DeVries, T J, et al. Pathology, Research and Practice 192: 718-733,1996.

17. Kubota, S., et al. International J. Cancer 70: 106-111, 1997.

18. Powell, W C and L M Matrisian. Current Topics in Microbiology andImmunology 213:1-21, 1996.

19. Seiki, M. Current Topics in Microbiology and Immunology 213: 23-32,1996.

20. Byers S W, C L Sommers, B. Hoxter, A M Mercurio and A J Tozeren.Cell Science 108: 2053-2064, 1995.

21. Yoneda, J., et al. Experimental Cell Research 217: 169-179, 1995.

22. Tsang, T C, et al. Cancer Research 54: 882-886, 1994.

23. Grant D S, et al. Pathology, Research and Practice. 190: 854-863,1994.

24. Spiegel, S., et al. Breast Cancer Research and Treatment 312:337-348, 1994.

25. Thorgeirsson, U P, et al. J. Neuro-Oncology 18: 89-103, 1993.

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What is claimed is:
 1. A method of inhibiting tumor invasion ormetastasis of a tumor comprising tumor cells which express RAGE in asubject, which comprises administering to the subject a therapeuticallyeffective amount of a form of human soluble Receptor for AdvancedGlycation Endproduct (sRAGE)(SEQ ID NO:1).
 2. A method of inhibitingtumor invasion or metastasis of a tumor comprising tumor cells whichexpress RAGE in a subject, which comprises administering to the subjecta therapeutically effective amount of a form of human soluble Receptorfor Advanced Glycation Endproduct (sRAGE) comprising a peptide havingthe sequence from methionine at position 1 to glycine at position 22 ofSEQ ID NO:2 linked to alanine at position 1 and ending with isoleucineat position 98 of SEQ ID NO:1.
 3. The method of claim 1 or 2, whereinthe administration is intralesional, intraperitoneal, intramuscular orintravenous injection, infusion, intrathecal, subcutaneous,liposome-mediated delivery, topical, nasal, oral, ocular or oticdelivery.
 4. The method of claim 1 or 2, wherein the therapeuticallyeffective amount is administered hourly, daily, weekly, monthly, yearly,or one time.
 5. The method of claim 1 or 2, wherein the human sRAGE isadmixed with a pharmaceutically acceptable carrier.
 6. The method ofclaim 1 or 2, wherein the therapeutically effective amount comprises adose of from about 100 ng/day/kg body weight of the subject to about 200mg/day/kg body weight of the subject.